Numerous epidemiologic and animal studies analyzing the impact of reproductive events such as puberty, pregnancy, and parity on early events in carcinogenesis suggest that the developmental state of the breast plays a critical role in the determination of breast cancer risk (Lambe et al., N. Engl. J. Med., 331:5-9 (1994); MacMahon et al., Bull. WHO, 43:209-221 (1970); MacMahon et al., Int. J. Cancer, 29:13-16 (1982); Newcomb et al., N. Engl. J. Med., 330:81-87 (1994); Russo et al., J. Natl. Cancer Inst., 61:1439-1449 (1978); Russo et al., Lab. Invest., 57:112-137 (1987)). In fact, a woman's lifetime risk of developing breast cancer is intrinsically related to reproductive events, particularly those that affect the differentiated state of the breast. Results from both human epidemiology and animal model systems indicate that an early first full-term pregnancy results in a permanent change in the breast that confers a decreased risk for the subsequent development of breast cancer (Medina et al., J. Natl. Cancer Inst., 91:967-969 (1999); Russo et al., Breast Cancer Res. Treat., 2:5-73, (1982); Russo et al., J. Natl. Cancer Inst., 61:1439-1449 (1978); MacMahon et al., 1970). This implies an intrinsic relationship between the process of carcinogenesis and normal pathways of differentiation and development in the breast.
The findings that aborted pregnancies, the majority of which occur prior to the third trimester, are not protective against breast cancer and that lactation has only a minimal protective effect compared with full-term pregnancy suggest that parity-induced protection against breast cancer results from physiological changes that occur late in pregnancy (Michels et al., Cancer Causes Control, 6:75-82 (1995); Melbye et al., N. Engl. J. Med., 336:81-85 (1997)). As a result, the protective effect of parity has been hypothesized to result from the impact of terminal differentiation on the susceptibility of the mammary epithelium to carcinogenesis (Russo et al., 1982; Russo et al., 1978). Nevertheless, the molecular and cellular basis for this phenomenon is unknown. As such, understanding the developmental changes that occur in the breast late in pregnancy is essential for understanding the protected state of the breast associated with parity, particularly with respect to genes that control mammary proliferation and differentiation.
Protein kinases represent the largest class of genes known to regulate differentiation, development, and carcinogenesis in eukaryotes. Many protein kinases function as intermediates in signal transduction pathways that control complex processes such as differentiation, development, and carcinogenesis (Birchmeier et al., BioEssays, 15:185-190 (1993); Bolen, Oncogene, 8:2025-2031 (1993); Rawlings et al., Immunol. Rev., 138:105-119 (1994)). Accordingly, studies of protein kinases in a wide range of biological systems have led to a more comprehensive understanding of the regulation of cell growth and differentiation (Bolen 1993; Fantl et al., Annu. Rev. Biochem., 62:453-481(1993); Hardie, Symp. Soc. Exp. Biol., 44:241-255 (1990)).
Not surprisingly, several members of the protein kinase family have been reported to be involved in the pathogenesis of cancer both in humans and in rodent model systems (Cardiff et al., Cancer Surv., 16:97-113 (1993); Dickson et al., Cancer Treatment Res., 61:249-273 (1992); Guy et al., Genes Dev., 8:23-32 (1994); Guy et al., Proc. Natl. Acad. Sci. USA, 89:10578-10582 (1992); Slamon et al., Science, 244:707-712 (1989)). For instance, the EGF receptor and ErbB2/HER2 are each amplified and overexpressed in subsets of highly aggressive breast cancers, and these molecules may thereby provide prognostic information relevant to clinical treatment and outcome (Klijn et al., Cold Spring Harbor Laboratory Press, Vol. 18, pp. 165-198 (1993); Slamone et al., Science, 235, 177-182 (1987); Slamon et al., 1989). Furthermore, overexpression of specific protein kinases, or of ligands for protein kinases, in the mammary epithelium of transgenic animals results in neoplastic transformation (Cardiff et al., 1993); Guy et al., 1994); Muller et al., Cell, 54:105-115 (1988) Muller et al., EMBO J., 9:907-913 (1990)).
One particular family of protein kinases, the Ca2+/calmodulin-dependent (CaM) kinases are known to regulate cellular processes as diverse as neurotransmitter release, muscle contraction, cell cycle control, transcriptional regulation, metabolism, and gene transcription (Fukunaga et al., Jpn. J. Pharmacol., 79:7-15 (1999); Matthews et al., Mol. Cell. Biol., 14:6107-6116 (1994); Polishchuk et al., FEBS Lett., 362:271-275 (1995); Schulman, Curr. Opin. Cell Biol., 5:247-253 (1993); Sheng et al., Science, 252:1427-1430 (1991)). For example, point mutations in the Drosophila calmodulin gene result in defects in development including pupal lethality and ectopic wing vein formation (Nelson et al., Genetics, 147:1783-1798 (1997)). Furthermore, calmodulin expression is regulated during cardiac development, and overexpression of calmodulin in murine cardiomyocytes results in cardiomyocyte hypertrophy (Gruver et al., Endocrinology, 133:376-388 (1993)). Like calmodulin, it has been reported that CaM kinases play diverse roles in development including CaMKIV in T-cell maturation and CaMKII in cell cycle regulation (Lukas et al., San Diego: Academic Press 65-168 (1998); Nairn et al., Semin. Cancer Biol., 5:295-303 (1994); Hanissian et al., J. Biol. Chem., 268:20055-20063 (1993); Krebs et al., Biochem. Biophys. Res. Commun., 241:383-389(1997)).
Ca2+ is an important intracellular second-messenger molecule in eukaryotic signal transduction pathways. Many of the effects of Ca2+ are mediated through its interaction with the Ca2+-binding protein, calmodulin. The Ca2+/calmodulin complex is, in turn, required for maximal activation of CaM-dependent protein kinases. In addition, as a family, CaM kinases share structural and functional homology both in the kinase catalytic domain and in a regulatory region composed of composite autoinhibitory and CaM binding domains (Hanks et al., Methods Enzymol., 200:38-79 (1991); Hanks et al., Science, 241:42-52 (1988); Haribabu et al., EMBO J., 14:3679-3686 (1995); Knighton et al., Science, 258:130-135 (1992); Picciotto et al., I. Adv. Pharmacol., 36:251-275 (1996); Yokokura et al., J. Biol. Chem., 270:23851-23859 (1995)).
Despite these similarities, significant differences exist between CaM kinase family members. For instance, this family includes members with high substrate specificity, such as myosin light-chain kinase (MLCK) and phosphorylase kinase, as well as members with broader substrate specificities collectively referred to as the multifunctional CaM kinases, such as CaMKI, CaMKIV, and members of the CaMKII subfamily (Braun et al., Annu. Rev. Physiol., 57: 417-445 (1995); Cawley et al., J. Biol. Chem., 268:1194-1200 (1993); Herring et al., J. Biol. Chem., 265:1724-1730 (1990); Matthews et al., 1994; Schulman, 1993). Other properties that differ among CaM kinase family members include their subcellular localization, regulation by autophosphorylation, and regulation by other proteins. In addition, CaM kinases have unique amino- and carboxyl-terminal domains that contribute to kinase-specific differences in subcellular localization, subunit interactions, and other protein-protein interactions. Much of the information available regarding the multifunctional CaM kinases is derived from studies conducted in the brain, where they are expressed at high levels.
In light of these findings, it is clear that until the present invention, there has remained a need to identify and study the role of protein kinases in postnatal development and carcinogenesis, as well as provide insight into how the decision to proliferate or differentiate is made in mammary epithelial cells. Moreover, identification of cancer-linked protein expression product, and the gene encoding same, offers previously unavailable diagnostic and therapeutic solutions to carcinogenesis.